Gamma three protease

ABSTRACT

Gamma three protease is provided, a novel aspartyl class protease that is capable of taking part in the processing of amyloid precursor protein (APP) to Aβ peptide. Gamma three protease may be involved in the development and/or progression of Alzheimers disease. Methods of identifying inhibitors of gamma three protease, useful in the prevention or treatment of Alzheimers disease, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/311,410, filed Aug. 10, 2001, the contents of which are incorporatedherein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention is directed to the field of Alzheimer's disease.In particular, the present invention provides a novel aspartyl proteaseinvolved in the processing of Alzheimer's precursor protein to theβ-amyloid peptide.

BACKGROUND OF THE INVENTION

Alzheimer's disease is a common, chronic neurodegenerative disease,characterized by a progressive loss of memory and sometimes severebehavioral abnormalities, as well as an impairment of other cognitivefunctions that often leads to dementia and death. It ranks as the fourthleading cause of death in industrialized societies after heart disease,cancer, and stroke. The incidence of Alzheimer's disease is high, withan estimated 2.5 to 4 million patients affected in the United States andperhaps 17 to 25 million worldwide. Moreover, the number of sufferers isexpected to grow as the population ages.

A characteristic feature of Alzheimer's disease is the presence of largenumbers of insoluble deposits, known as amyloid plaques, in the brainsof those affected. Autopsies have shown that amyloid plaques are foundin the brains of virtually all Alzheimer's patients and that the degreeof amyloid plaque deposition correlates with the degree of dementia(Cummings & Cotman, 1995, Lancet 326:1524–1587). While some opinionholds that amyloid plaques are a late stage by-product of the diseaseprocess, the consensus view is that amyloid plaques are more likely tobe intimately, and perhaps causally, involved in Alzheimer's disease.

A variety of experimental evidence supports this view. For example, Aβ,a primary component of amyloid plaques, is toxic to neurons in cultureand transgenic mice that overproduce Aβ in their brains show significantdeposition of Aβ into amyloid plaques and significant neuronal toxicity(Yankner, 1990, Science 250:279–282; Mattson et al., 1992, J. Neurosci.12:379–389; Games et al., 1995, Nature 373:523–527; LaFerla et al.,1995, Nature Genetics 9:21–29). Mutations in the APP gene, leading toincreased Aβ production, have been linked to heritable forms ofAlzheimer's disease (Goate et al., 1991, Nature 349:704–706;Chartier-Harlan et al., 1991, Nature 353:844–846; Murrel et al., 1991,Science 254:97–99; Mullan et al., 1992, Nature Genetics 1:345–347).Presenilin-1 (PS1) and presenilin-2 (PS2) related familial early-onsetAlzheimer's disease (FAD) shows disproportionately increased productionof Aβ1-42, the 42 amino acid isoform of Aβ, as opposed to Aβ1-40, the 40amino acid isoform (Scheuner et al, 1996, Nature Medicine 2:864–870).The longer isoform of Aβ is more prone to aggregation than the shorterisoform (Jarrett et al, 1993, Biochemistry 32:4693–4697). Injection ofthe insoluble, fibrillar form of Aβ into monkey brains results in thedevelopment of pathology (neuronal destruction, tau phosphorylation,microglial proliferation) that closely mimics Alzheimer's disease inhumans (Geula et al., 1998, Nature Medicine 4:827–831). See Selkoe,1994, J. Neuropathol. Exp. Neurol. 53:438–447 for a review of theevidence that amyloid plaques have a central role in Alzheimer'sdisease.

Aβ, a 39–43 amino acid peptide derived by proteolytic cleavage of theamyloid precursor protein (APP), is the major component of amyloidplaques (Glenner & Wong, 1984, Biochem. Biophys. Res. Comm.120:885–890). APP is actually a family of polypeptides produced byalternative splicing from a single gene. Major forms of APP are known asAPP₆₉₅, APP₇₅₁, and APP₇₇₀, with the subscripts referring to the numberof amino acids in each splice variant (Ponte et al., 1988, Nature331:525–527; Tanzi et al., 1988, Nature 331:528–530; Kitaguchi et al.,1988, Nature 331:530–532). APP is membrane bound and undergoesproteolytic cleavage by at least two pathways. In one pathway, cleavageby an enzyme known as α-secretase occurs while APP is still in thetrans-Golgi secretory compartment (Kuentzel et al., 1993, Biochem J.295:367–378). This cleavage by α-secretase occurs within the Aβ portionof APP, thus precluding the formation of Aβ. In another proteolyticpathway, cleavage of the Met₆₇₁-Asp₆₇₂ bond (numbered according to the751 amino acid protein) by an enzyme known as β-secretase occurs. Thiscleavage by β-secretase generates the N-terminus of Aβ. The C-terminusis formed by cleavage by a second enzyme known as γ-secretase. TheC-terminus is actually a heterogeneous collection of cleavage sitesrather than a single site since γ-secretase activity occurs over a shortstretch of APP amino acids rather than at a single peptide bond.Peptides of 40 or 42 amino acids in length (Aβ1-40 and Aβ1-42,respectively) predominate among the C-termini generated by γ-secretase.Aβ1-42 is more prone to aggregation than Aβ1-40, is the major componentof amyloid plaque (Jarrett et al., 1993, Biochemistry 32:4693–4697; Kuoet al., 1996, J. Biol. Chem. 271:4077–4081), and its production isclosely associated with the development of Alzheimer's disease (Sinha &Lieberburg, 1999, Proc. Natl. Acad. Sci. USA 96:11049–11053). The bondcleaved by γ-secretase appears to be situated within a transmembranedomain of APP. It is unclear as to whether the C-termini of Aβ1-40 andAβ1-42 are generated by a single γ-secretase protease with sloppyspecificity or by two distinct proteases. For a review that discussesAPP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447–453.

Much interest has focused on the possibility of inhibiting thedevelopment of amyloid plaques as a means of preventing or amelioratingthe symptoms of Alzheimer's disease. To that end, a promising strategyis to inhibit the activity of β- and γ-secretase, the two enzymes thattogether are responsible for producing Aβ. This strategy is attractivebecause, if the formation of amyloid plaques as a result of thedeposition of Aβ is a cause of Alzheimer's disease, inhibiting theactivity of one or both of the two secretases would intervene in thedisease process at an early stage, before late-stage events such asinflammation or apoptosis occur. Such early stage intervention isexpected to be particularly beneficial (see, e.g., Citron, 2000,Molecular Medicine Today 6:392–397).

To that end, various assays have been developed that are directed to theidentification of compounds that may interfere with the production of Aβor its deposition into amyloid plaques. U.S. Pat. No. 5,441,870 isdirected to methods of monitoring the processing of APP by detecting theproduction of amino terminal fragments of APP. U.S. Pat. No. 5,605,811is directed to methods of identifying inhibitors of the production ofamino terminal fragments of APP. U.S. Pat. No. 5,593,846 is directed tomethods of detecting soluble Aβ by the use of binding substances such asantibodies. Esler et al., 1997, Nature Biotechnology 15:258–263described an assay that monitored the deposition of Aβ from solutiononto a synthetic analogue of an amyloid plaque. The assay was suitablefor identifying compounds that could inhibit the deposition of Aβ.However, this assay is not suitable for identifying substances, such asinhibitors of γ-secretase, that would prevent the formation of Aβ. Thus,the assay of Esler is directed to a step that is further along in theformation of amyloid plaque than is the assay described in thisapplication.

Various groups have cloned and sequenced cDNA encoding a protein that isbelieved to be β-secretase (Vassar et al., 1999, Science 286:735–741;Hussain et al., 1999, Mol. Cell. Neurosci. 14:419–427; Yan et al., 1999,Nature 402:533–537; Sinha et al., 1999, Nature 402:537–540; Lin et al.,2000, Proc. Natl. Acad. Sci. USA 97:1456–1460) but the identity ofγ-secretase has been more elusive. A pair of proteins known aspresenilin-1 and presenilin-2 are viewed as possible candidates (Selkoe& Wolfe, 2000, Proc. Natl. Acad. Sci. USA 97:5690–5692).

Presenilin-1 (PS1) and presenilin-2 (PS2) are polytopic membraneproteins that are involved in γ-secretase-mediated processing of APP.The most common cause of familial early-onset Alzheimer's disease is theautosomal dominant inheritance of assorted mutations in the PSi gene(Sherrington et al., 1995, Nature 375:754–760). These PSi mutations leadto increased production of Aβ1-42 (Scheuner et al., 1996, NatureMedicine 2:864–870; Duff et al., 1996, Nature 383:710–713; Borchelt etal., 1996, Neuron 17:1005–1013). Similarly, certain mutations in PS2cause familial early-onset Alzheimer's disease and increased generationof Aβ1-42 (Levy-Lahad et al., 1995, Science 269:970–973). Culturedisolated neurons from PS1-deficient mice exhibit reducedγ-secretase-mediated cleavage of APP (De Strooper et al., 1998, Nature391:387–390). It was suggested that PS1 might influence trafficking ofAPP and/or γ-secretase or it might play a more direct role inproteolytic cleavage of APP. Directed mutagenesis of two conservedtransmembrane-situated aspartates in PS1 was shown to inactivateγ-secretase activity in cellular assays, suggesting that PS1 is either arequired diaspartyl cofactor for γ-secretase or is itself γ-secretase,an intramembranous aspartyl protease (Wolfe et al., 1999, Nature398:513–517). Moreover, Li et al., 2000, Nature 405:689–694 madephotoactivatable derivatives of a highly specific and potent aspartylprotease transition state analog inhibitor and found that the inhibitorselectively labeled presenilin fragments.

Despite results such as those described above, it is still uncertainwhether PS1 and PS2 are responsible for the γ-secretase activity that isrelevant to the processing of APP in connection with Alzheimer'sdisease. It is desirable to identify all the proteases that may haveγ-secretase activity and thus may be involved in the development ofAlzheimer's disease. Therefore, the identification and purification ofnovel proteins possessing γ-secretase activity is valuable. Theavailability of such novel proteases would allow for the development ofassays to discover inhibitors of such proteases. Such inhibitors arelikely to be valuable in the treatment of Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention is directed to γ3 protease, a novel aspartyl classprotease that is capable of taking part in the processing of amyloidprecursor protein (APP) to Aβ peptide. Since the deposition of Aβ in thebrains of patients suffering from Alzheimer's disease is believed toplay an important role in the development of this disease, γ3 proteasemay be involved in the development and/or progression of Alzheimer'sdisease. Therefore, inhibitors of γ3 protease may have utility in theprevention or treatment of Alzheimer's disease. Methods of identifyingsuch inhibitors are disclosed.

Also disclosed are membrane preparations containing partially purifiedγ3 protease as well as methods of further purifying γ3 protease andidentifying cDNA encoding γ3 protease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that L-685,458 is able to essentially completely blockγ-secretase activity at pH 7.0 (the pH optimum for γ-secretase activityassociated with presenilin-1/presenilin-2) but that significantγ-secretase activity remains at pH 6.0 (the pH optimum for γ-secretaseactivity associated with γ3 protease). The source of γ3 protease was amembrane preparation from HeLa S3 cells prepared as in Example 9. Theproduction of the γ3 protease substrate and the conditions for cleavageare described in Example 10. The procedure for determining whether thesubstrate had been cleaved by γ3 protease was as in Example 6.

FIG. 2A shows the pH dependency of the generation of Aβ peptide having alength of 40 amino acids (Aβ1-40) in the presence of 1 μM of L-645,458,a concentration sufficient to essentially completely inhibit theγ-secretase activity associated with presenilins 1 and 2. The presenceof γ3 protease activity having a pH optimum of about 6 is clearly seen.FIG. 2B shows the pH dependency of the generation of Aβ peptide having alength of 42 amino acids (Aβ1-42) in the presence of 1 μM of L-645,458,a concentration sufficient to essentially completely inhibit theγ-secretase activity associated with presenilins 1 and 2. Again, thepresence of γ3 protease activity having a pH optimum of about 6 isclearly seen. The source of γ3 protease was ES-PBD18 membranes. ES-PBD18membranes were prepared from wild-type mouse embryonic stem cellsaccording to the procedure of Example 11.

FIG. 3A shows that pepstatin A inhibits the production of Aβ1-40 by γ3protease. FIG. 3B shows that pepstatin A inhibits the production ofAβ1-42 by γ3 protease.

FIG. 4A–B shows the cDNA sequence (SEQ.ID.NO.:1) and FIG. 4C shows theamino acid sequence (SEQ.ID.NO.:2) of the 695 amino acid splice variantof wild-type Alzheimer's precursor protein (APP). See GenBank accessionno. Y00264 and Kang et al., 1987, Nature 325:733–736.

FIG. 5 shows a schematic representation of the HTRF method of detectiondescribed herein. In this embodiment, the first antibody is either 4G8,6E10, or WO-2. The second antibody is either G2-10 (which specificallydetects Aβ1-40) or G2-11 (which specifically detects Aβ1-42). Theepitopes recognized by these antibodies are shown in FIG. 6. The firstantibody is coupled to XL-665 by a streptavidin/biotin link. Specificsignal generation, indicating γ3 protease cleavage of the substrate,occurs when Europium cryptate (EuK)-coupled G2-10 or G2-11 (secondantibodies) and the first antibody-biotin-streptavidin-XL-665 pair arebrought into proximity as a result of binding to the Aβ peptide. Thisresults in fluorescence resonance energy transfer (FRET) from EuK toXL-665.

FIG. 6 shows the epitopes recognized by the monoclonal antibodies ofFIG. 5. The solid bars indicate the amino acid sequences against whichthe monoclonal antibodies were raised, or, where determined, theirrecognition epitopes (Kim et al., 1988, Neurosci. Res. Comm. 2:121–130;Kim et al., 1990, Neurosci. Res. Comm. 7:113–122; Ida et al., 1996, J.Biol. Chem. 271:22908–22914). The human and rodent Aβ sequences areshown with the three amino acid differences between them underlined inthe rodent sequence. The major cleavage sites of the APP molecule by thevarious secretases are indicated by arrows: β=β secretase; α=αsecretase; γ=γ secretase and γ3 protease.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention:

“Substances” can be any substances that are generally screened in thepharmaceutical industry during the drug development process. Forexample, substances may be low molecular weight organic compounds (e.g.,having a molecular weight of less than about 2,000 daltons andpreferably less than about 1,000 daltons), RNA, DNA, antibodies,peptides, or proteins.

The conditions under which substances are employed in the methodsdescribed herein are conditions that are typically used in the art forthe study of protein-ligand interactions: e.g., salt conditions such asthose represented by such commonly used buffers as PBS or in tissueculture media; a temperature of about 4° C. to about 55° C.; incubationtimes of from several seconds to several hours. The pH can be a pH atwhich the activity of γ3 protease can be distinguished from the activityof γ-secretase, e.g., a pH of about 5.8 to 6.2. Alternatively, the pHcan be closer to pH 7.0, a pH at which γ-secretase is active, if themethods are run in the presence of a γ-secretase inhibitor.

The present invention relates to a novel, membrane-bound aspartyl classprotease, γ3 protease. γ3 protease is an aspartyl class protease sinceits activity is inhibited by pepstatin A. γ3 protease cleavesAlzheimer's precursor protein (APP), as well as artificial substratesincorporating portions of APP, at the same or similar sites asγ-secretase. Since cleavage of APP by γ-secretase is thought to be anessential step in the generation of Aβ peptide, γ3 protease may also beinvolved in the generation of Aβ. Thus, γ3 protease, like γ-secretase,may play a role in the development and/or progression of Alzheimer'sdisease.

The activity of γ3 protease can be distinguished from the known γsecretase activity involving presenilin-1 and presenilin-2 by thefollowing characteristics:

γ3 protease activity migrates with an M_(r) of approximately 60 kDa to120 kDa during gel filtration analysis while the corresponding M_(r) ofthe γ secretase complex containing presenilin-1 is approximately 2×10⁶kDa.

γ3 protease activity is not susceptible to inhibition by L-685,458 whilethe activity of the γ secretase complex containing presenilin-1 orpresenilin-2 is inhibited by L-685,458.

γ3 protease activity displays a pH optimum of 6.0 while the activity ofthe γ secretase complex containing presenilin-1 or presenilin-2 displaysa pH optimum of 6.8.

γ3 protease activity is present in presenilin-1/presenilin-2 doubleknockout cells.

γ3 protease proteolytic products are dominated by Aβ1-42, which is moreprone to aggregate and form Aβ plaques than Aβ1-40.

The present invention provides assays for γ3 protease. In broad terms,such assays comprise providing a source of γ3 protease, incubating theγ3 protease in the presence of a suitable substrate under suitableconditions such that the γ3 protease can cleave the substrate intoproduct, and determining the presence and/or the amount of productproduced from the substrate by the γ3 protease.

The source of the γ3 protease may be a preparation of purified protein,but can also be cells that express γ3 protease, or membranes from suchcells. In certain embodiments, the cells recombinantly express γ3protease. That is, they have been transfected with an expression vectorencoding γ3 protease such that the expression vector directs theexpression of γ3 protease in the cells. Preferably, the recombinantcells do not naturally express γ3 protease, or at least, express γ3protease to only an insignificant level as compared to the levelachieved via recombinant expression.

A variety of host cells are suitable for recombinant expression of γ3protease. Particularly preferred are mammalian cell lines. In particularembodiments, the test cells and control cells are selected from thegroup consisting of: L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCCCCL 1.2), HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I(ATCC CRL 1616), BS-C-1 (ATCC CCL 26), T24 (ATCC HTB4), and MRC-5 (ATCCCCL 171).

In preferred embodiment, the source of γ3 protease is cells derived frompresenilin-1/presenilin-2 double knockout mice. These mice have had boththeir presenilin-1 and presenilin-2 genes disrupted so that cells fromthese mice cannot express functional presenilin-1 or presenilin-2. Thus,these cells have no γ-secretase activity that is due to presenilin-1 orpresenilin-2.

The source of γ3 protease can be suitable membrane preparations fromcells that express γ3 protease. Such membrane preparations can beobtained by procedures, such as that described in Example 1, comprising:

(a) lysis of the cells;

(b) low speed centrifugation to remove unbroken cells and largesubcellular structures;

(c) high speed centrifugation of the supernatant from step (b) to pelletmembranes containing γ3 protease;

(d) resuspension of the membranes from step (c);

(e) assaying the resuspended membranes of step (d) for γ3 proteaseactivity.

In particular embodiments, low speed centrifugation is carried out bycentrifuging at from about 750×g to about 1,500×g; preferably from about900×g to about 1,200×g; and most preferably at about 1,000×g at atemperature of about 2° C. to about 10° C., preferably from about 3° C.to about 6° C., and most preferably at about 4° C. for about 5 minutesto about 20 minutes, preferably for about 8 minutes to about 15 minutes,and most preferably for about 10 minutes. The low speed centrifugationstep may be repeated if desired.

In particular embodiments, high speed centrifugation is carried out bycentrifuging from about 75,000×g to about 150,000×g; preferably fromabout 90,000×g to about 120,000×g; and most preferably at about100,000×g at a temperature of about 2° C. to about 10° C., preferablyfrom about 3° C. to about 6° C., and most preferably at about 4° C. forabout 30 minutes to about 90 minutes, preferably for about 50 minutes toabout 75 minutes, and most preferably for about 60 minutes.

In particular embodiments, resuspension of the membranes is carried outby use of a Douce Homogenizer.

Solubilized, purified γ3 protease can be obtained from such membranepreparations. Recovery of soluble γ3 protease activity may be achievedusing a zwitterionic detergent to extract the membrane preparationfollowed by suitable steps to isolate the solubilized γ3 protease.

Accordingly, the present invention includes a method of purifying γ3protease comprising:

(a) preparing membranes containing 3 protease;

(b) solubilizing the membranes in a zwitterionic detergent;

(c) centrifuging the solubilized membranes to obtain a supernatant;

(d) passing the supernatant over an affinity column to bind γ3 proteasein the supernatant to the affinity column;

(e) eluting γ3 protease from the affinity column.

In particular embodiments, the amount of zwitterionic detergent is about1% to about 2% (w/v). Examples of zwitterionic detergents include CHAPSOand CHAPS.

In particular embodiments, the affinity column is a Pepstatin A affinitycolumn.

In particular embodiments, the centrifuging is carried out bycentrifuging from about 75,000×g to about 150,000×g; preferably fromabout 90,000×g to about 120,000×g; and most preferably at about100,000×g at a temperature of about 2° C. to about 10° C., preferablyfrom about 3° C. to about 6° C., and most preferably at about 4° C. forabout 30 minutes to about 90 minutes, preferably for about 50 minutes toabout 75 minutes, and most preferably for about 60 minutes. A suitableinstrument for such centrifugation is a BECKMAN XL-90 Ultracentrifuge.

Examples of cells expressing γ3 protease include HeLa S3, humanembryonic kidney (HEK293) cells and Chinese hamster ovary (CHO) cells.Preferred cells producing γ3 protease are presenilin-1(PS-1)/presenilin-2 (PS-2) double knockout cells that have both copiesof both PS-1 and PS-2 disabled. These PS-1/PS-2 double knockout cellsthus have no PS-1 and PS-2 activity. Such cells can be derived fromembryonic stem cells obtained from the embryos of the founders fromcross breeding transgenic mice (PS1 knock-out and PS2 knock-out mice).

γ3 protease recognizes and cleaves substrates that have the amino acidsequence of APP in the vicinity of the γ-secretase cleavage sites. ForAPP695, these γ-secretase cleavage sites are predominately betweenpositions 635 and 636 (to produce (Aβ1-40) or between positions 637 and638 (to produce (Aβ1-42). Shown immediately below is SEQ.ID.NO.:3, whichconsists of the relevant portions of human APP (SEQ.ID.NO.:2), beginningat position 1 of Aβ. The predominant γ3 protease cleavage sites areindicated by arrows.

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAII (SEQ. ID. NO.: 3)         ↓  ↓ GLMVGGVVIA TVIVITLVMLKKKQYTSIHH GVVEVDAAVTPEERHLSKMQQNGYENPTYKFF EQMQN

Cleavage at the left arrow (between positions 40 and 41 of SEQ.ID.NO.:3)gives rise to Aβ1-40 while cleavage at the right arrow (betweenpositions 42 and 43 of SEQ.ID.NO.:3) gives rise to Aβ1-42.

Suitable substrates of γ3 protease include polypeptides comprising anamino acid sequence from the vicinity of these cleavage sites. Forexample, suitable substrates would include polypeptides comprising allof SEQ.ID.NO.:3; polypeptides comprising positions 10–70 ofSEQ.ID.NO.:3; polypeptides comprising positions 15–65 of SEQ.ID.NO.:3;polypeptides comprising positions 20–60 of SEQ.ID.NO.:3; polypeptidescomprising positions 25–55 of SEQ.ID.NO.:3; polypeptides comprisingpositions 30–50 of SEQ.ID.NO.:3; polypeptides comprising positions 31–49of SEQ.ID.NO.:3; polypeptides comprising positions 32–48 ofSEQ.ID.NO.:3; polypeptides comprising positions 33–47 of SEQ.ID.NO.:3;polypeptides comprising positions 34–46 of SEQ.ID.NO.:3; polypeptidescomprising positions 35–45 of SEQ.ID.NO.:3; and polypeptides comprisingpositions 36–44 of SEQ.ID.NO.:3.

Corresponding polypeptides from versions of APP from mammals other thanhumans are also suitable substrates. Shown immediately below isSEQ.ID.NO.:4, which consists of the relevant portion of APP fromrodents, e.g., rats and mice.

DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAII (SEQ. ID. NO.: 4)         ↓  ↓ GLMVGGVVIA TVIVITThis rodent sequence differs from the human sequence at the threeunderlined residues.

The substrate for γ3 protease may be full-length APP, either wild-typeor one of the various mutants forms of APP, or some subset of the aminoacids of APP that contains the γ-secretase cleavage sites. A cDNAsequence encoding full length wild-type APP is shown in FIG. 4A–B and isSEQ.ID.NO.:1 while the amino acid sequence of full length wild-type APPis shown in FIG. 4C and is SEQ.ID.NO.:2. APP may be provided byrecombinantly expressing cDNA encoding APP in suitable host cells andpurifying the APP by methods well known in the art. Alternatively, APPmay be produced by in vitro coupled transcription/translation systemsthat are also well known in the art.

APP may also be provided as a γ3 protease substrate withoutpurification. Cells expressing APP, either naturally or throughrecombinant means, are used directly, by exposing such cells tosubstances that are to be tested in the assays of the present inventionwithout first purifying or isolating APP from the cells. The productionof suitable γ3 protease cleavage products from the APP in the cells isthen measured or detected.

In certain embodiments of the assays of the present invention, where theassay system is expected to contain γ-secretase activity associated withpresenilin-1 or presenilin-2, it is advantageous to run the assay underconditions that inhibit the presenilin-1 or presenilin-2-associatedγ-secretase activity. Such conditions include running the assay in thepresence of an inhibitor of the presenilin-1/presenilin-2-associatedγ-secretase activity (e.g., L-685,458) and/or running the assay at a pHof about 6.0 (a pH at which the activity ofpresenilin-1/presenilin-2-associated γ-secretase is minor but which isoptimum for γ3 protease).

In certain embodiments, the inhibitor of thepresenilin-1/presenilin-2-associated γ-secretase activity is atransition-state analogue inhibitor of γ-secretase. Esler et al., 2000,Nature Cell Biol. 2:428–434 disclose a series of such inhibitors,including methods of synthesis of the inhibitors. The structure of thisseries of inhibitors is shown in Example 13. The disclosures of Esler etal., 2000, Nature Cell Biol. 2:428–434 are incorporated herein byreference, in their entirety.

Another series of transition-state analogue inhibitors is described inInternational Patent Publication WO 01/53255. This series includes theinhibitor L-685,458 (see Example 8). Representative members of thisseries are shown in Example 14. L-685,458 is also disclosed in Shearmanet al., 2000, Biochemistry 39:8698–8704. The disclosures ofInternational Patent Publication WO 01/53255 and Shearman et al., 2000,Biochemistry 39:8698–8704 are incorporated herein by reference, in theirentirety.

A particular embodiment of the assays of the present invention is amethod of identifying inhibitors of the activity of γ3 protease. Such amethod comprises:

(a) incubating:

-   -   (i) a source γ3 protease;    -   (ii) a substrate of γ3 protease:

in the presence and in the absence of a substance;

(b) determining whether the substrate has been cleaved by the γ3protease;

where, if the substrate has been cleaved by γ3 protease to a lesserextent in the presence as compared to the absence of the substance, thenthe substance is an inhibitor of γ3 protease.

The method of identifying inhibitors of γ3 protease can be used toscreen libraries of substances or other sources of substances toidentify substances that are inhibitors of the activity of γ3 protease.Such identified inhibitory substances can serve as “leads” for thedevelopment of pharmaceuticals that can be used to treat patients havingAlzheimer's disease.

In certain embodiments, the assays of the present invention employ anartificial substrate derived from APP. One particular version of such aγ3 protease substrate is a fusion protein comprising a carboxy-terminalfragment of APP (a “β-CTF domain”) and a hydrophilic polypeptide moiety.The β-CTF domain provides a polypeptide that can be cleaved by γ3protease activity. The hydrophilic polypeptide moiety allows for theβ-CTF domain to be cleaved by detergent-solubilized γ3 protease bypromoting substrate solubility. The β-CTF domain approximates theC-terminal fragment of APP after cleavage by β-secretase or is afunctional derivative thereof.

SEQ.ID.NO.:5 provides an example of a β-CTF domain. SEQ.ID.NO.:5 is anaturally occurring sequence corresponding to the β-CTF portion of APP(amino acids 596–695) along with an N-terminus methionine. TheN-terminus methionine facilitates recombinant production of thesubstrate. SEQ.ID.NO.:5 is as follows:

MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI (SEQ. ID. NO.: 5)IGLMVGGVVIATVIVITLVMLKKKQYTSIHHG VVEVDAAVTPEERHLSKMQQNGYENP TYKFF EQMQN

The second component of the γ3 protease substrate, the hydrophilicpolypeptide moiety, is preferably chosen to increase the solubility ofthe γ3 protease substrate in a zwitterionic detergent. Hydrophilicmoieties can be obtained by taking into account the known charges andpolarity of different amino acid R groups. Preferably, the presence ofthe hydrophilic moiety does not result in a substrate having asubstantial decrease in activity.

Different embodiments concerning the overall length and charge of thehydrophilic moiety are provided as follows: in different embodimentsconcerning the length, the length is about 5 to about 20 amino acids,about 8 to about 12 amino acids, or about 8 amino acids; in differentembodiments concerning the overall charge, the charge is greater than±2, ±3, or ±4. With respect to a negative charge, a greater chargeindicates a higher negative charge value.

Preferably, the hydrophilic moiety comprises, consists essentially of,or consists of, a polypeptide substantially identical to SEQ.ID.NO.:6:DYKDDDDK. Substantially identical to SEQ.ID.NO.:6 indicates that withina corresponding 8 amino acid stretch (no gaps) there is a two, one, orzero amino acid difference. Preferably, the hydrophilic moiety consistsof the amino acid sequence of SEQ.ID.NO.:6.

In an embodiment of the present invention, the γ3 protease substratecomprises, consists essentially of, or consists of, a sequencesubstantially similar to SEQ.ID.NO.:7. SEQ.ID.NO.:7 corresponds toSEQ.ID.NO.:5 along with a carboxyl terminal SEQ.ID.NO.:6 sequence.Preferably, the γ3 protease substrate comprises, consists essentiallyof, or consists of SEQ.ID.NO.:7.

MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI (SEQ. ID. NO.: 7)IGLMVGGVVIATVIVITLVMLKKKQYTSIHHG VVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNDYKDDDDK

Based on the disclosure provided herein γ3 protease substrates can beproduced using standard biochemical synthesis and recombinant nucleicacid techniques. Techniques for chemical synthesis of polypeptides arewell known in the art. (See, for example, Vincent, in Peptide andProtein Drug Delivery, New York, N.Y., Dekker, 1990). Recombinantsynthesis techniques for polypeptides are also well known in the art.Such techniques employ a nucleic acid template for polypeptidesynthesis. Starting with a particular amino acid sequence and the knowndegeneracy of the genetic code, a large number of different encodingnucleic acid sequences can be obtained that encode the desired γ3protease substrate.

Recombinant synthesis of polypeptides that are γ3 protease substratescan be achieved in a host cell using an expression vector. An expressionvector contains recombinant nucleic acid encoding a desired polypeptidealong with regulatory elements for proper transcription and processing.Generally, the regulatory elements that are present in an expressionvector include a transcriptional promoter, a ribosome binding site, aterminator, and an optionally present operator. A preferred element is apolyadenylation signal providing for processing in eukaryotic cells.Other preferred elements include an origin of replication for autonomousreplication in a host cell, a selectable marker, a limited number ofuseful restriction enzyme sites, and a potential for high copy number.Examples of expression vectors are cloning vectors, modified cloningvectors, specifically designed plasmids, and viruses.

A variety of expression vectors can be used. Commercially availableexpression vectors which are suitable include, but are not limited to,pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3,pcDNA3.1, pCR3.1 (Invitrogen, San Diego, Calif.), EBO-pSV2-neo (ATCC37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pCI.neo (Promega), pTRE(Clontech, Palo Alto, Calif.), pV1Jneo, pIREsneo (Clontech, Palo Alto,Calif.), pCEP4 (Invitrogen, San Diego, Calif.), pSC11, and pSV2-dhfr(ATCC 37146). The choice of vector will depend upon cell type in whichit is desired to express the γ3 protease substrates, as well as on thelevel of expression desired, and the like.

Nucleic acid encoding a polypeptide that is a γ3 protease substrate canbe expressed in a cell without the use of an expression vectoremploying, for example, synthetic mRNA or native mRNA. Additionally,mRNA can be translated in various cell-free systems such as wheat germextracts and reticulocyte extracts, as well as in cell based systems,such as frog oocytes. Introduction of mRNA into cell based systems canbe achieved, for example, by microinjection.

Techniques for introducing nucleic acid into an appropriate environmentfor expression, for expressing the nucleic acid to produce γ3 proteasesubstrate, and for isolating expressed γ3 protease substrate are willknown in the art. Examples of such techniques are provided in referencessuch as Ausubel, Current Protocols in Molecular Biology, John Wiley,1987–1998, and Sambrook et al., in Molecular Cloning, A LaboratoryManual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989.

The γ3 protease substrate can be employed in assays measuringmembrane-bound or detergent-solubilized γ3 protease. Production ofcleavage products can be detected by Aβ peptide or hydrophilic moietyproduct formation.

Assay conditions employing membrane-bound or detergent-solubilized γ3protease allow for detectable γ3 protease activity. Such conditionsinclude an effective amount of a zwitterionic detergent, a buffer, andan appropriate temperature.

An effective amount of a particular zwitterionic detergent results indetectable cleavage. Suitable detergents and amounts can be determinedby evaluating the effect of a particular detergent on γ3 proteaseactivity. Preferred zwitterionic detergents present during the assay areCHAPS and CHAPSO. A preferred percentage of such detergents is about0.1% to about 0.5%.

An example of a reaction condition allowing for γ3 protease activity isprovided as follows: 1.7 μM substrate incubated with cell membranes ordetergent solubilized γ3 protease in the presence of 0.25% CHAPSO inbuffer (50 mM MES, pH 6.0, 5 mM MgCl₂, 5 mM CaCl₂, 150 mM KCl) at 37° C.Based on the present disclosure such reaction conditions can be alteredto provide a wide range of additional reaction conditions allowing forγ3 protease activity. Preferably, changes to the reaction conditions donot result in a substantial decrease in activity.

γ3 protease activity can be stopped using techniques well known in theart for stopping enzymatic reactions. Preferably, γ3 protease activityis stopped using reagents compatible with subsequent analysis.

Cleavage of γ3 protease substrates can be measured by detectingformation of an Aβ type product or a product containing the hydrophilicmoiety. The presence of either of these products can be measured usingtechniques such as those employing antibodies and radioactive,electrochemiluminescent, or fluorescent labels. If needed or desirable,a purification step enriching the different products may be employed.Examples of purification steps include the use of antibodies, separationgels, and columns. Preferably, substrate cleavage by γ3 protease isassayed for by detecting the presence of Aβ1-40 or Aβ1-42.

A method for detecting substrate cleavage via the detection of Aβ1-40 orAβ1-42 is described in Clarke & Shearman, 2000, J. Neurosci. Meth.102:61–68. This methods involves the use of a homogeneous time-resolvedfluorescence assay (HIRF). A first antibody that recognizes a central orN-terminal portion of Aβ1-40 and Aβ1-42 is conjugated to XL-665, whichserves as a fluorescence acceptor. A second antibody, that specificallyrecognizes either Aβ1-40 or Aβ1-42, is conjugated to Europium cryptate,which serves as a fluorescent donor. The second antibody does notrecognize the intact substrate but rather recognizes a neoepitope thatis formed by cleavage of the substrate by γ3 protease. When γ3 proteasecleaves the substrate, the neoepitope is formed, thus permitting bindingof the second antibody. When the second antibody is bound, the Europiumcryptate fluorescent donor and the XL-665 flourescent acceptor arebrought close enough together that fluorescence resonance energy tranfer(FRET) can occur between the Europium cryptate and the XL-665. This FRETcan be measured by suitable instrumentation and can serve as a measureof substrate cleavage by γ3 protease. FIGS. 5 and 6 illustrate theconcepts behind this method.

Another method for product detection, employing electrochemiluminescencewith a capture antibody and an antibody specific for either Aβ1-40 orAβ1-42, is described in Example 6 below. This method was used to obtainthe data shown in FIGS. 1–3.

The following non-limiting examples are presented to better illustratethe invention.

EXAMPLE 1

Purification of γ3 Protease-Containing Membranes for Use in Assays

-   -   1. Resuspend PS1/PS2 double KO cell pellets (1 ml packed cell        volume, ˜2×10⁸ cells) in 5 ml cold MES buffer (50 mM MES, 5 mM        MgCl₂, 5 mM CaCl₂, 150 mM KCl, pH 6.0 and 1× protease inhibitor        cocktails)    -   2. Pass through French press at 300 psi; repeat one more time.        Always wash the French press cylinder with cold H₂O two or three        times before using and pass cold buffer through before use. When        finished, wash with H₂O until the drainage is clear.    -   3. Centrifuge the broken cell homogenate at 1,000×g at 4° C. for        10 min. Collect supernatant.    -   4. Resuspend the pellet from step 3 in 5 ml of fresh buffer        using a Douce Homogenizer.    -   5. Disrupt the membrane pellet with a Dounce homogenizer (10        strokes)    -   6. Centrifuge at 1,000×g at 4° C. for 10 min. Collect        supernatant.    -   7. Combine supernatants from steps 3 and 6.    -   8. Centrifuge the combined supernatants at 100,000×g at 4° C.        for 1 hr    -   9. Discard the supernatant from step 8 and resuspend the pellet        in 2 ml of buffer (50 mM MES, 5 mM MgCl₂, 5 mM CaCl₂, 150 mM        KCl, pH 6.0 and 1× protease inhibitor cocktails) and centrifuge        the resuspended pellet at 100,000×g at 4° C. for 1 hr.    -   10. Discard the supernatant from step 9 and resuspend the pellet        in 2 ml of buffer (50 mM MES, 5 mM MgCl₂, 5 mM CaCl₂, 150 mM        KCl, pH 6.0 and 1× protease inhibitor cocktails) and homogenize        with 5 strokes of a Dounce homogenizer.    -   11. The prepared membranes were then aliquoted and can be stored        at −70° C.

EXAMPLE 2

Purification of γ3 Protease

Membranes prepared as in Example 1 (e.g., from PS1 and PS2 double knockout mouse cells or HeLa cells) at a protein concentration of 2.5 mg/mlin buffer A (50 mM 2-[N-Morpholino]ethane-sulfonic acid (MES), pH 6.0, 5mM MgCl₂, 5 mM CaCl₂, 150 mM KCl, containing “complete” proteaseinhibitor cocktail (Boehringer Mannheim, Indianapolis, Ind.) aresolubilized by treatment with 1% CHAPSO for 60 min at 4° C. andcentrifugation at 100,000×g for 60 min. Solubilized proteins arecentrifuged at 40,0000 rpm for 1 hr. Supernatant is removed and assayedfor enzyme activity. The supernatant is then further diluted 4-fold withMES buffer (50 mM, pH 6.0), resulting in final concentration of 0.25%CHAPSO. A Pepstatin A affinity column is prepared from pepstatin Aimmobilized agarose (Cat# P-2032, Sigma) and pre-equilibrated withloading buffer (MES 50 mM, pH 6.0) and the supernatant is loaded on thecolumn. The column is washed with three column volumes of loadingbuffer. Elution buffer (20 mM Tris buffer, pH 7.5) is applied on thecolumn to elute the γ3 protease. The activity of the enzyme is monitoredwith an in vitro assay. Active fractions are pooled and concentrated onCentracon-10,000.

EXAMPLE 3

PS-1/PS-2 Double Knockout Cells

Such cells can be derived from embryonic stem (ES) cells obtained fromthe embryos of the founders from cross breeding transgenic mice (PS1knock-out and PS2 knock-out mice). ES cells were cultured in 90% DMEM,10% CALF serum, with 1% Penicillin/Streptomycin medium at 37° C., 5%CO₂, humidified incubator.

Methods of preparing PS-1/PS-2 double knockout cells are described inHerreman et al., 2000, Nature Cell Biol. 2:461–462.

EXAMPLE 4

Cloning of γ3 Protease

Isolated γ3 protease prepared according to Example 2 is run out on anSDS-PAGE gel and the purity of the enzyme is evaluated by staining withCoomassie blue. A preparative gel is prepared and the γ3 proteaseprotein bands are cut out and analyzed by peptide mapping. Sequenceinformation is then used for searching protein data bases to identifyentries corresponding to γ3 protease. Upon obtaining the sequence of theprotein from the databases, degenerate oligonucleotide primers aredesigned and used for PCR against specific tissue cDNA libraries. Theamplified DNA bands are sequenced and full-length cDNA clones obtained.DNA encoding γ3 protease is then cloned into suitable host cells tooverexpress the protein. Large scale purification can then be carriedout to obtain enough protein for further characterization.

EXAMPLE 5

Recombinant production of the γ3 protease substrate

A DNA fragment encoding amino acids 596–695 of the 695 amino acidisoform of APP (APP695) and SEQ.ID.NO.:6 at the C-terminus was generatedby PCR amplification of APP695 cDNA using appropriate primers. Theemployed primers had the following sequences:

GGAATTCCATATGGATGCAG AATTCCGACA (SEQ. ID. NO.: 8) TG; andCGCGGATCCCTATTTATCGTCATCGTCTTTG (SEQ. ID. NO.: 9)TAGTCGTTCTGCATCTGCTCAAAGAACTTG

The Met that serves as the translation start site is residue 596 ofAPP695 (the P1 residue with respect to the γ3 protease cleavage site).This DNA fragment was inserted into the procaryotic expression vectorpET2-21b (Novagen, Madison, Wis.). The recombinant protein ofSEQ.ID.NO.:7 was overproduced in E. coli [strain BL21(DE3)] and purifiedby Mono-Q column chromatography (Pharmacia Biotech).

SEQ.ID.NO.:10 provides a nucleic acid sequence encoding the recombinantprotein of SEQ.ID.NO.:7 along with a stop codon.

ATGGATGCAGAATTCCGACATGACTCAGGATA (SEQ. ID. NO.: 10)TGAAGTTCATCATCAAAAATTGGTGTTCTTTG CAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGC GACAGTGATCGTCATCACCTTGGTGATGCTGAAGAAGAAACAGTACACATCCATTCATCATGGT GTGGTGGAGGTTGACGCCGCTGTCACCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACG GCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACGACTACAAAGACGATGACGA TAAATAG

EXAMPLE 6

Detection of the Aβ Peptide Products of γ3 Protease Activity byElectrochemiluminescence (ECL)

Aβ peptides were detected using a sandwich assay employing an antibodyto capture the peptide and an antibody to detect the presence of thepeptide. Detection was achieved by using electrochemiluminescence (ECL)(Yang et al., 1994, Bio/Technology 12:193–194; Khorkova et al., 1998, J.Neurosci. Meth. 82:159–166) and an Origen 1.5 Analyzer (Igen Inc.,Gaithersburg, Md.).

Capture was performed using the 4G8 murine monoclonal antibody (SenetekPLC, Maryland Heights, Mo.). The 4G8 murine monoclonal antibody binds anepitope in the Aβ peptide (about amino acids 18–21 of SEQ.ID.NO.:3) thatis immediately distal to the α-secretase cleavage site. The 4G8monoclonal antibody was biotinylated with Biotin-LC-Sulfo-NHS-Ester(Igen Inc., Gaithersburg, Md.).

Detection was achieved using the G2-10 murine monoclonal antibody andthe FCA3542 rabbit antibody. The G2-10 murine monoclonal antibody(provided by K. Beyreuther, University of Heidelberg, Germany) binds theC-terminus that is exposed after γ3 protease-mediated cleavage togenerate amino acid 40 of the Aβ1-40 peptide. (Ida et al., 1996, J.Biol. Chem. 271:22908–22914). The FCA3542 rabbit antibody (provided byF. Checler, IPMC du CNRS, Valbonne, France) binds the C-terminus that isexposed after γ3 protease-mediated cleavage to generate amino acid 42 ofthe Aβ1-42 peptide. (Barelli et al., 1997, Mol. Med. 3:695–707.)

The G2-10 and FCA3542 antibodies were ruthenylated with TAG-NHS Ester(Igen Inc., Gaithersburg, Md.). Aβ(x-40) was detected with biotinylated4G8 and ruthenylated G2-10. Aβ(x-42) was detected with biotinylated 4G8and ruthenylated FCA3542.

EXAMPLE 7

γ3 Protease Assay Using Artificial Substrate

In vitro assays measuring γ3 protease activity are performed using γ3protease that is isolated by the method described in Example 2.Alternatively, membranes prepared as in Example 1 may be used.SEQ.ID.NO.:7 substrate (1.7 μM) is incubated with isolated γ3 protease(50 nM) in presence of detergent in buffer B (50 mM MES, pH 6.0, 5 mMMgCl₂, 5 mM CaCl₂, 150 mM KCl) at 37° C. Generally, 0.25% CHAPSO isprovided as the detergent. The reactions are stopped by adding RIPA (150mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris HCl, pH 8.0). Thesamples are centrifuged at 1,000 g for 1 min and the supernatantsolutions are assayed for the Aβ peptides by ECL. The Aβ1-40- andAβ1-42-related products from γ3 protease-mediated processing ofSEQ.ID.NO.:7 substrate possess a Met at the N-terminus and are thusdefined as M-Aβ1-40 and M-Aβ1-42, respectively.

EXAMPLE 8

L-685,458

L-685,458 is a γ-secretase inhibitor having the following structure:

L-685,458 contains an hydroxyethylene dipeptide isostere and is thoughtto function as a transition state analog mimic of aspartyl proteases(Shearman et al., 2000, Biochemistry 39:8698–8704). L-685,458 wasprepared as follows:{1S-Benzyl-4-R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester (L-685,458) was prepared by the coupling of2R-benzyl-5S-tert-butoxycarbonylamino-4R-(tert-butyldimethylsilanyloxy)-6-phenylhexanoicacid (Evans et al., 1985, J. Org. Chem. 50:4615–4625) with Leu-Phe-NH2followed by deprotection with tetrabutylammonium fluoride. The synthesisof{1S-benzyl-4-R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2S-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester (L-682,679) has been described previously (DeSolms et al., 1991, J. Med. Chem. 34:2852–2857).{1S-Benzyl-4-R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2-oxo-5-phenylpentyl}carbamicacid tert-butyl ester (L-684,414) was prepared by pyridiniumdichromate-mediated oxidation of L-682,679.

EXAMPLE 9

Membranes Containing γ3 Protease from HeLa Cells

HeLa S3 cells from American Type Culture Collection (Rockville, Md.)were grown in bioreactors (Analytical Biological Services; Wilmington,Del.) in 90% DMEM, 10% fetal bovine serum, 2 mM glutamine and 100 μg/mleach of penicillin and streptomycin. Frozen HeLa S3 cells wereresuspended in buffer A (50 mM 2-[N-Morpholino]ethane-sulfonic aid, MES)MES, pH 6.0, 5 mM MgCl₂, 5 mM CaCl₂, 150 mM KCl) containing “complete”protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, Ind.).The cells were broken by single-pass through a French Press (SpectronicInstruments, Rochester, N.Y.). Cell debris and nuclei were removed bycentrifugation at 800×g for 10 min. The supernatant solutions werecentrifuged at 100,000×g for 60 min. The ensuing pellets wereresuspended by Dounce homogenizer in buffer A and the centrifugation wasrepeated. The final membrane pellets were resuspended by Doucehomogenizer in buffer A to yield a protein concentration ofapproximately 12 mg/ml. All procedures were performed at 4° C. Themembranes were stored at −70° C.

EXAMPLE 10

Production of γ3 Protease Substrate and Conditions for Cleavage by γ3Protease

A DNA fragment encoding amino acids 596–695 of the 695 amino acidisoform of APP (APP695) and the Flag sequence (DYKDDDDK (SEQ.ID.NO.:6)at the C-terminus was generated by PCR amplification withsuitably-designed oligonucleotides and the APP695 cDNA. The Met thatserves as the translation start site is residue 596 of APP695 (the P1residue with respect to the β-secretase cleavage site). This DNAfragment was inserted into the procaryotic expression vector pET2-21b(Novagen, Madison Wis.). The recombinant protein, C100Flag(SEQ.ID.NO.:7), was overproduced in E. coli [strain BL21(DE3)] andpurified by Mono-Q column chromatography (Pharmacia Biotech). C100Flag(1.7 μM) was incubated with cell membranes (0.5 mg/ml) in presence ofCHAPSO, CHAPS or Triton X-100 (0, 0.125, 0.25, 0.5, or 1%) in buffer B(50 mM 1,4-piperazinediethanesulfonic acid (PIPES), pH 7.0, 5 mM MgCl₂,5 mM CaCl₂, 150 mM KCl) at 37° C. The reactions were stopped by addingRIPA (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mMTris HCl, pH 8.0) and boiling for 5 min. The samples were centrifugedand the supernatant solutions were assayed for the Aβ peptides byelectrochemiluminescence (ECL). The Aβ1-40- and Aβ1-42-related productsfrom γ3 protease-mediated processing of C100Flag possess a Met at theN-terminus and are thus defined as M-Aβ1-40 and M-Aβ1-42, respectively.Supernatant solution from CHAPSO-extracted HeLa cell membranes(solubilized γ3 protease) was incubated with C100Flag in buffer Bcontaining 0.25% CHAPSO and subsequently assayed for M-Aβ1-40 andM-Aβ1-42 using ECL.

EXAMPLE 11

Production of Membranes Containing γ3 Protease from Wild-Type MouseEmbryonic Stem Cells

-   1. Resuspend pellet (2–10 ml, ˜10⁸–10⁹ cells) in 2× volume of cold    MES buffer (50 mM (2-[N-Morpholino]ethane-sulfonic aid (MES), 5 mM    MgCl₂, 5 mM CaCl₂,150 mM KCl, pH 6.0) plus 1× Protease Inhibitor    Cocktail (#1836153, complete Mini protease inhibitor cocktail,    Roche).-   2. Homoginize pellet using Douce homogenizer at 4° C., 20 times.-   3. Spin at “2000” rpm, 4° C., 10 min. Collect supernatant.-   4. Resuspend pellet in 5–10 ml fresh cold MES buffer using Douce    homogenizer, homogenize 10×, spin at 2000 rpm, 4° C., 10 min.    Collect supernatant.-   5. Combine supernatant from step 3 and 4, balance, ultra centrifuge    at 35K, 4° C., 1 hr.-   6. Discard the supernatant, resuspend pellet in 5 ml MES buffer    using Dounce homogenizer, ultra centrifuge at 35K, 4° C., 30 min.-   7. Discard supernatant, resuspend pellet in 1–3 ml MES buffer,    homogenize 5×, check protein concentration, and save in liquid    nitrogen.

EXAMPLE 12

Gel Exclusion Chromatography of γ3 Protease

Gel exclusion chromatography was performed as follows: solubilized γ3protease was diluted 4-fold in buffer (50 mM PIPES, pH 7.0, 5 mM MgCl₂,5 mM CaCl₂, 150 mM KCl) and 1 ml was loaded onto a Superose 6 HR 10/30column (Pharmacia Biotech) using an AKTAexplorer chromatography system(Pharmacia Biotech). The column was eluted with buffer B containing0.25% CHAPSO. Fractions (0.5 ml) were analyzed for in vitro γ3 proteaseactivity as described herein.

EXAMPLE 13

Transition-State Analogue Inhibitors γ-Secretase Activity

Esler et al., 1997, Nature Biotechnology 15:258–263 disclose theγ-secretase inhibitors 1, BrA-1,1-Bt, and BrA-1-Bt, the structures ofwhich are shown below.

-   1 R₁=t-Bu-O (tert-butoxy); R₂═CO₂Me-   BrA-1 R₁═BrCH₂; R₂═CO₂Me-   1-Bt R₁=t-Bu-O (tert-butoxy); R₂═CH₂O-Bt (Bt=biotin)-   BrA-1-Bt R₁═BrCH₂; R₂═CH₂O-Bt (Bt=biotin)

EXAMPLE 14

Additional Transition-State Analogue Inhibitors γ-Secretase Activity

The following γ-secretase inhibitors are disclosed in InternationalPatent Publication WO 01/53255. Example 7 is L-685,458.

Stucture Example Number

Example 1

Example 2

Example 3

Example 4

Example 5

Example 6

Example 7

Example 8

Example 9

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A membrane preparation from eukaryotic cells containing γ3 proteasehaving an M_(r) of approximately 60 kDa to 120 kDa during gel filtrationanalysis where the γ3 protease is catalytically active and has anactivity that: is not susceptible to inhibition by L-685,458; issusceptible to inhibition by Pepstatin A; displays a pH optimum of 6.0;cleaves a substrate having an amino acid sequence comprisingSEQ.ID.NO.:3 between positions 40 and 41 of SEQ.ID.NO.:3 or betweenpositions 42 and 43 of SEQ.ID.NO.:3; where the membrane preparation hasbeen prepared by a method comprising: (a) lysis of cells expressing γ3protease to produce a lysate; (b) low speed centrifugation of the lysateto form a pellet and a supernatant from the lysate; (c) high speedcentrifugation of the supernatant from step (b) to form a pellet and asupernatant from the supernatant from step (b); and (d) resuspension ofthe pellet from step (c) to form the membrane preparation; and (e)determining that the membrane preparation contains catalytically activeγ3 protease by incubating the membrane preparation in the presence of asuitable substrate of γ3 protease under suitable conditions such thatthe γ3 protease cleaves at least a portion of the substrate into productand identifying product produced from the substrate by the γ3 protease.2. The membranes of claim 1 where the eukaryotic cells arepresenilin-1/presenilin-2 double knockout cells or HeLa cells.
 3. Themembranes of claim 1 where low speed centrifugation is carried out bycentrifuging at from about 750×g to about 1,500×g at a temperature ofabout 2° C. to about 10° C. for about 5 minutes to about 20 minutes andhigh speed centrifugation is carried out by centrifuging at from about75,000×g to about 150,000×g at a temperature of about 2° C. to about 10°C. for about 30 minutes to about 90 minutes.
 4. The membranes of claim 1where low speed centrifugation is carried out by centrifuging at about1,000×g at a temperature of about 4° C. for about 10 minutes and highspeed centrifugation is carried out by centrifuging at about 100,000×gat a temperature of about 4° C. to about 60 minutes.
 5. The membranes ofclaim 1 where step (e) is carried out in the presence of an inhibitor ofγ-secretase or at a pH of about 5.8 to 6.2.
 6. Purified γ3 proteaseprepared by a method comprising: (a) solubilizing the membranes of claim1 in a zwitterionic detergent; (b) centrifuging the solubilizingmembranes to obtain a supernatant; (c) passing the supernatant over anaffinity column to bind γ3 protease in the supernatant to the affinitycolumn; (d) eluting γ3 protease from the affinity column.
 7. Thepurified γ3 protease of claim 6 where the zwitterionic detergent isCHAPS or CHAPSO at a concentration of about 1% to 2% (w/v) and theaffinity column is a Pepstatin A affinity column.
 8. A method ofpreparing a membrane suspension containing γ3 protease comprising: (a)lysis of cells expressing γ3 protease to produce a lysate; (b) low speedcentrifugation of the lysate to form a pellet and a supernatant from thelysate; (c) high speed centrifugation of the supernatant from step (b)to form a pellet; (d) resuspension of the pellet from step (c) to formthe membrane suspension; (e) determining that the membrane suspensionfrom step (d) contains catalytically active γ3 protease is by incubatingthe membrane suspension in the presence of a suitable substrate of γ3protease under suitable conditions such that the γ3 protease cleaves atleast a portion of the substrate into product and identifying productproduced from the substrate by the γ3 protease; where the γ3 proteasehas an M_(r) of approximately 60 kDa to 120 kDa during gel filtrationanalysis and has an activity that: is not susceptible to inhibition byL-685,458; is susceptible to inhibition by Pepstatin A; displays a pHoptimum of 6.0; cleaves a substrate having an amino acid sequencecomprising SEQ.ID.NO.:3 between positions 40 and 41 of SEQ.ID.NO.:3 orbetween positions 42 and 43 of SEQ.ID.NO.:3.
 9. The method of claim 8where step (e) is carried out in the presence of an inhibitor ofγ-secretase or at a pH of about 5.8 to 6.2.
 10. An assay for γ3 proteasecomprising: (a) providing a source of γ3 protease; (b) incubating the γ3protease in the presence of a suitable substrate under suitableconditions such that the γ3 protease cleaves the substrate into product;and (c) determining the amount of product produced from the substrate bythe γ3 protease.
 11. The assay of claim 10 where step (b) is carried outin the presence of an inhibitor of γ-secretase or at a pH of about 5.8to 6.2.
 12. A method of identifying an inhibitor of γ3 proteasecomprising: (a) incubating: (i) a source γ3 protease; (ii) a substrateof γ3 protease: in the presence and in the absence of a substance; (b)determining whether the substrate has been cleaved by the γ3 protease;where, if the substrate has been cleaved by γ3 protease to a lesserextent in the presence as compared to the absence of the substance, thenthe substance is an inhibitor of γ3 protease.
 13. The method of claim 12where the source of γ3 protease is a membrane preparation containing γ3protease.
 14. The method of claim 13 where the membrane preparationcomprises membranes isolated from eukaryotic cells, the membranescontaining γ3 protease having an M_(r) of approximately 60 kDa to 120kDa during gel filtration analysis where the γ3 protease iscatalytically active and has an activity that: is not susceptible toinhibition by L-685,458; is susceptible to inhibition by Pepstatin A;displays a pH optimum of 6.0; cleaves a substrate having an amino acidsequence comprising SEQ.ID.NO.:3 between positions 40 and 41 ofSEQ.ID.NO.:3 or between positions 42 and 43 of SEQ.ID.NO.:3.
 15. Themethod of claim 12 where the substrate of γ3 protease is a polypeptidecomprising all of SEQ.ID.NO.:3; a polypeptide comprising positions 10–70of SEQ.ID.NO.:3; a polypeptide comprising positions 15–65 ofSEQ.ID.NO.:3; a polypeptide comprising positions 20–60 of SEQ.ID.NO.:3;a polypeptide comprising positions 25–55 of SEQ.ID.NO.:3; a polypeptidecomprising positions 30–50 of SEQ.ID.NO.:3; a polypeptide comprisingpositions 31–49 of SEQ.ID.NO.:3; a polypeptide comprising positions32–48 of SEQ.ID.NO.:3; a polypeptide comprising positions 33–47 ofSEQ.ID.NO.:3; a polypeptide comprising positions 34–46 of SEQ.ID.NO.:3;a polypeptide comprising positions 35–45 of SEQ.ID.NO.:3; or apolypeptide comprising positions 36–44 of SEQ.ID.NO.:3.
 16. The methodof claim 12 where the substrate comprises an amino acid sequenceselected from the group consisting of: SEQ.ID.NO.:2, SEQ.ID.NO.:3,SEQ.ID.NO.:4, SEQ.ID.NO.:5, and SEQ.ID.NO.:7.
 17. The method of claim 13where the γ3 protease: has an M_(r) of approximately 60 kDa to 120 kDaduring gel filtration analysis: is not susceptible to inhibition byL-685,458; is susceptible to inhibition by Pepstatin A; displays a pHoptimum of 6.0; cleaves a substrate having an amino acid sequencecomprising SEQ.ID.NO.:3 between positions 40 and 41 of SEQ.ID.NO.:3 orbetween positions 42 and 43 of SEQ.ID.NO.:3.
 18. The method of claim 12where step (a) is carried out in the presence of an inhibitor ofγ-secretase or at a pH of about 5.8 to 6.2.
 19. The method of claim 18where the inhibitor of γ-secretase is selected from the group consistingof: L-685,458, 1, BrA-1, 1-Bt, and BrA-1-Bt.